Our long-term goal is to generate cell-based cancer vaccines for the treatment of established metastatic cancer, particularly metastatic breast cancer. Because CD4+ T lymphocytes are critical cells for optimal cell-mediated immunity, we have focused on making vaccines that activate tumor-specific CD4+ T cells. The vaccines are based on two hypotheses: 1) Tumor cells genetically modified to express syngeneic MHC class II, co-stimulatory, and T cell activation molecules directly present tumor-encoded antigens to CD4+ T cells; 2) Activation of tumor-specific CD4+ T cells facilitates activation of tumor-specific CD8+ T cells and results in potent anti-tumor immunity and long-term memory. Mechanistic and therapeutic studies support both hypotheses, and in a separate project we are translating this approach to the clinic. Although the vaccines have significant therapeutic efficacy against large burdens of widely disseminated, spontaneous metastatic cancer, some mice are non-responders and although survival time for others is significantly extended, most mice still die. As we adapt the vaccines to the clinic, it is highly desirable to continue developing new approaches in innovative animal models, so that we can constantly input improved strategies to the translational studies. Further development is dependent on understanding the underlying mechanisms by which the vaccines stimulate immunity. Studies, therefore, have also focused on examining basic aspects of antigen presentation by the vaccines. To further improve and test the vaccines in mice, the following therapeutic and mechanistic questions will be addressed: 1) Do the MHC class Il-based vaccines protect very high risk mice from developing mammary cancer? 2) Can expression of transcriptional regulatory elements down-regulate Invariant chain and convert MHC class II+Ii+DM+ tumor cells to vaccines? 3) How do the cell-based vaccines present MHC class 11-restricted, endogenously synthesized tumor antigens? 4) What is the role of the vaccine cells in induction of anti-tumor immunity? Which effector cells, cytokines, and chemokines are induced during therapy? 5) Does tumor burden correlate with tumor-induced immuno-suppression? Does surgical removal of primary tumor reverse mmuno-suppression?